Composition, methods and devices useful for manufacturing of implantable articles

ABSTRACT

The application is directed to aqueous dispersible biodegradable compositions of esters which are the condensation reaction product of a polyol and a diacid which are within a matrix of hydrated polypeptide. The compositions are useful in additive manufacturing and other applications for use with implantable articles. In some embodiments, the ester in the compositions is the product of a glyercol-sebacic acid condensation reaction.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of, and priority to, U.S. App. Nos.62/037,425 filed Aug. 14, 2014 and 62/037,640 filed Aug. 15, 2014 bothof which are hereby incorporated by reference in their entirety.

FIELD

This application relates to the field of material science and materialsuseful in the manufacture of implantable articles, such as additivemanufacturing. More specifically, the present invention provides anaqueous dispersible biodegradable composition of esters formed from apolyol/diacid condensation reaction within a matrix of hydratedpolypeptide.

BACKGROUND

A number of pressing problems confront the healthcare industry. As ofJune 2012 the United Network for Organ Sharing (UNOS) had 114,636patients registered as needing an organ transplant. According to UNOS,between January and March 2012 only 6,838 transplants were performed.Each year more patients are added to the UNOS list than transplants areperformed, resulting in a net increase in the number of patients waitingfor a transplant.

This has placed an economic burden on the US healthcare economy both indirect and indirect costs. Organ transplant and regenerative medicineare expected to eventually meet the challenge of replacement andregeneration as the portfolio of biomaterials increases. In the past 50years, scientists have been limited in the choice of biomaterials usefulin tissue/organ engineering.

One new biomaterial, poly(glycerol sebacate), was developed for tissueengineering and has expanded into therapeutic areas including drugdelivery, orthopedics, cardiovascular, neurovascular and soft tissuerepair. With benefits such as little to no fibrous capsule formation,antimicrobial activity, non-immunogenicity, among others, the materialis desirable for implantable as well as topical applications. Currentforms of this biomaterial, however, have some limitations on its abilityto be manipulated into various forms for specific applications.

Additive manufacturing has become an important tissue scaffoldfabrication tool in tissue engineering and regenerative medicine.Precise patient specific 3-D tissue scaffold constructs when designed bysoftware imported from such 3-D medical diagnostic imaging modalities asMRI and ultrasound, can reconstruct in vitro and in vivo tissueframework (tissue scaffold) from subject tissue.

There is a serious limitation to the choice of biocompatible andresorbable polymer technologies to build the basic compositionssupporting scaffold structures by additive manufacturing. Historically,lactide and glycolide polymers have been the resorbable polymers ofchoice. While the basic chemistry is resorbable, the question ofbiocompatibility has been challenged. Once in the wound space, lactideand glycolide polymers break down, releasing highly acidic by-productswhich extend the healing period, adversely impact the immune system, andform scar tissue, disrupting the return of native function.

SUMMARY

Exemplary embodiments are directed to aqueous dispersible biodegradablecompositions of esters which are the condensation reaction products of apolyol and a diacid and are within a matrix of hydrated polypeptide, aswell as to articles made therefrom and methods and devices which employthose compositions.

According to an exemplary embodiment, a composition comprises water, anester of a polyol and a diacid and a polypeptide. In some embodiments,the ester comprises a glyercol-sebacic acid ester compound, such as apolymeric glyercol-sebacic acid ester compound

According to another embodiment, a method for forming a compositioncomprises mixing a solid polypeptide in water to form a polypeptidehydrogel and adding a glyercol-sebacic acid ester compound to thepolypeptide hydrogel.

According to another embodiment, a method for printing athree-dimensional article comprises extruding a first two-dimensionallayer of the compositions described herein onto a substrate and buildinga second two-dimensional layer of that composition upon the first layer.

According to yet another embodiment, a method for forming an articlecomprises extruding a fiber of the compositions described herein, whichin some embodiments involves co-extrusion with one or more otherpolymeric compositions in forming the fiber.

According to still another embodiment, a print head for use in additivemanufacturing comprises a nozzle and a plurality of reservoirs, eachreservoir containing a biocompatible material, the nozzle comprising atip in fluid communication with each of the reservoirs via a feed line,the nozzle having a pitched bore to accomplish mixing of thebiocompatible materials during extrusion.

Among the advantages of exemplary embodiments is that compositions areprovided that can overcome many of the limitations of conventionalmaterials used in implantable articles, such as limiting scarring andproviding a compliance modulus that better approximates native tissue.

Another advantage is that compositions are provided for use in formationof implantable materials that can result in regeneration of tissue inthe absence of exogenous factors like stem cells, mesenchymal cells,trophic agents or other biologics

Still another advantage is that compositions are provided thataccomplish biological benefits associated with its constituents in aform suitable for use in a variety of processes including additivemanufacturing, molding, coating, forming techniques, machining andextrusion.

Other features and advantages of the present invention will be apparentfrom the following more detailed description of exemplary embodimentsthat illustrate, by way of example, the principles of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of exemplary embodiments may bebetter understood when read in conjunction with the appended drawings.It should be understood, however, that the invention is not limited tothe precise arrangements and instrumentalities shown.

FIG. 1 is a 3-D printer assembly system in accordance with an exemplaryembodiment;

FIG. 2 is a lyophilized SEM of an enlarged cross sectional view of a 3-Dprinted structure in accordance with an exemplary embodiment;

FIG. 3 is a temperature sweep curve comparing a control solution to acomposition in accordance with an exemplary embodiment;

FIG. 4A is a top plan view of a nozzle according to an exemplaryembodiment of the invention;

FIG. 4B is a rear elevational view of the nozzle shown in FIG. 4A;

FIG. 4C is a perspective view of the nozzle shown in FIG. 4A;

FIG. 4D is a side view of the nozzle shown in FIG. 4A;

FIG. 4E is a nozzle in accordance with another embodiment;

FIG. 5A is a front cross sectional view of a nozzle according anexemplary embodiment of to the invention;

FIG. 5B is a front cross sectional view of the feedline assembly of thenozzle shown in FIG. 5A;

FIG. 5C is a side view of the nozzle shown in FIG. 5A;

FIG. 6A is a side elevation view of a heating element according to anexemplary embodiment of the invention;

FIG. 6B is a bottom elevational view of the heating element shown inFIG. 6A;

FIG. 6C is a front elevation view of the heating element shown in FIG.6A;

FIG. 6D is a perspective view of the heating element shown in FIG. 6A;

FIG. 7A is a bottom elevational view of the heating element shown inFIG. 6A assembled together with the nozzle shown in FIG. 4A;

FIG. 7B is a side elevation view of the heating element shown in FIG. 6Aassembled together with the nozzle shown in FIG. 4A; and

FIG. 7C is another side view of the heating element shown in FIG. 6Aassembled together with the nozzle shown in FIG. 4A.

FIG. 8A illustrates inhomogeneity in dehydrated, formed structures thatcan result when mixing steps during production are carried out in oneorder.

FIG. 8B illustrates homogeneity in dehydrated, formed structures and theimpact of PGS concentration on the aspect ratio when mixing steps duringproduction are carried out in a different order.

FIG. 9A is a collagen fiber shown at a magnification of ×500.

FIG. 9B is a 45 μm fiber of a composition in accordance with anexemplary embodiment of the present invention shown at a magnificationof ×500.

FIG. 10 is a thermally crosslinked and processed composition inaccordance with an exemplary embodiment showing consistent porosityacross its cross section and shown at a magnification of ×250.

FIG. 11A is a formed dehydrated composition in accordance with anexemplary embodiment in which the order of addition of mixing results inlarge pockets of glycerol-sebacic acid esters.

FIG. 11B is a formed dehydrated composition in accordance with anexemplary embodiment in which the order of addition of mixing results ina uniform distribution thereby creating a porous network.

FIG. 12 demonstrates the rheology and the impact glycerol-sebacic acidester concentration has on the storage modulus of compositions inaccordance with exemplary embodiments.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Exemplary embodiments are directed to aqueous dispersible biodegradablecompositions of esters which are the condensation reaction product of apolyol and a diacid (such as glycerol and sebacic acid) which are withina matrix of hydrated polypeptide. Exemplary embodiments also relate toarticles made from, and methods and devices which employ, thosecompositions, such as in additive manufacturing processes which are alsosometimes referred to as 3-D printing.

While compositions and other embodiments herein are primarily discussedwith respect to additive manufacturing processes, the invention is notso limited and the compositions may be employed for any other suitableapplication, including molding, coating, forming techniques, machiningor extrusion.

Bio-printing, such as of organs, tissues and other structures to beimplanted into the body, is a convergent technology of 3-D stereolithography, ink jet printing, tissue engineering, polymer chemistry andtissue-cell biology. The basic concept of 3-D organ printing in tissueengineering is to reproduce tissue, or tissue constructs, through theuse of formulations with cellular incorporation or the creation offreestanding scaffolds upon which cells can grow into organs.

3-D printing generally, also known as additive manufacturing, is wheresuccessive layers of material are laid down under computer control in anx-y plane to build the object layer by layer in the z-direction. Theprint-head nozzle can be an extrusion apparatus that deposits each layerof material in a 2-D horizontal plane like a common ink-jet printer.

A 3-D object can be sliced up into a finite number of horizontal planes.Reassembling these planes in the vertical direction recreates theobject. 3-D printing reassembles these planes to recreate the object. Inembodiments of the invention, instead of “ink” that is traditionallyused in 3-D printing, the 3-D print head nozzle extrudes a biocompatibleand/or bioresorbable polymer. Once a bioresorbable polymer compositionhas been deposited in a 2-D layer the print-head or platform stageindexes up or down (i.e., in the z-direction) wherein, the next 2-Dlayer is printed on the previous layer to rebuild the full structure.

Referring to FIG. 1, a 3-D printed object 10 is printed from acomposition 12. A 3-D printer assembly 100 includes a print head 14 anda platform 24. The print head 14 includes a nozzle 16 and a reservoir 18for holding composition 12. Print head 14 optionally further comprises aheating element 20. To print the 3-D printed object, the composition 12may be, in some embodiments, heated by the heating element 20, extrudedfrom reservoir 18 through tip 30 and deposited onto a substrate 22 toform a layer in the xy direction. The nozzle 16 and/or platform 24 maybe moved in the x, y, and z directions to enable deposition ofsuccessive xy layers, building the object 10 in the z direction. In someembodiments the 3-D printer assembly 100 further comprises a computer26. Computer 26 may be programmed to control movement of the nozzle 16and/or platform 24 as well as temperature of the heating element 20and/or platform 24.

While shown in FIG. 1 as being printed as discrete elements, it will beappreciated that a variety of manufacturing methods are contemplatedherein. In some embodiments, the composition 12 is extruded through thenozzle 16 in a continuous manner to form a fiber that can be used indirectly forming an article or wound for the production of yarn andsubsequent formation of textiles.

The composition 12 is a formulation of a bioresorbable polymer andpolypeptides and generally comprises water, a polypeptide, and acondensation polymer of a polyol and a diacid, such as aglycerol-sebacic acid ester compound, for example. In some embodiments,the composition 12 includes one or more of the bioresorbablebiocompatible materials based on metabolite building blocks disclosed inU.S. Pat. No. 7,722,894, which is hereby incorporated by reference inits entirety.

The glycerol-sebacic acid ester compound may be present in polymericform, having a molecular weight greater than 10,000 (also referred toherein as PGS); having a molecular weight of 10,000 or less, which mayconsidered an oligomeric form (also referred to herein as OGS); or somecombination of high and low molecular weight forms of the ester.

The use of PGS and OGS can overcome many of the limitations of lactidesand glycolides including exclusion of scarring. PGS is a bioelastomer,whereas the lactides and glycolides are rigid thermoplasticbioresorbable polymers. Additionally, the bioelastomeric featureprovides healing tissue with the appropriate compliance modulus thatencourages a scaffold environment more like native tissue. Aphysiologically compliant scaffold modulus emulates the extracellularmatrix (ECM) enhancing pro-healing cell signaling.

In some embodiments, the PGS of the composition 12 hydrolyzes (breaksdown) into cellular metabolites that may be consumed by the Krebs cycle,whereas the lactides and glycolides must be removed from the body bynon-metabolic processes. Without being bound by theory, when PGS is usedas an in vivo tissue scaffold, it may attract native tissue stem cellswithout the aid of exogenous trophic agents or progenitor cells.

In some embodiments, the composition 12 consists solely of water,polypeptide, and glycerol-sebacic acid ester compound to the exclusionof cells, biologics, trophic agents, growth agents, or other bioactivecompounds. Among the advantages of exemplary embodiments are thatregeneration of tissue may be accomplished in the absence of exogenousfactors like stem cells, mesenchymal cells, trophic agents or otherbiologics and that composition 12 may promote endogenous regeneration.It will be appreciated however, that such compounds are not excludedfrom composition 12 in all cases and that other embodiments may employone or more bioactives.

Without wishing to be bound by theory, it is believed that inembodiments which employ PGS in the composition 12, the composition'sbreakdown/erosion mechanism is as a surface eroder, which contrasts withmore conventional materials such as lactides and glycolides, which arebulk eroders. Surface erosion is attractive to both controlleddegradation of the biopolymer as a matrix scaffold material and as amatrix material for controlled drug or biologic release. Accordingly,the degradation mechanism will not lead to premature scaffold weakening.

In some embodiments, the composition 12 is prepared as a single uniformaqueous phase and is free of dispersing agents.

The composition 12 may be processed at extrusion temperatures compatiblewith living cells or described as “cold” extrusion, being in the rangeof about 35° C. to about 40° C. in some embodiments, which is a relativeterm with respect to generally higher polymer melt or flow temperatures(e.g. greater than about 200° C., for example). An improved “cold”extrusion nozzle 16 is provided which raises the nozzle temperatureslightly within body temperature range in order to increase thepotential for a higher solids polymer flow as well as form a smootherextrudate.

By the nature of 3-D printing, the material extruded must be able toflow through a nozzle as well as stick to the build platform or belowlayer in order to create an object. In typical Fused FilamentFabrication (FFF), this is done by melting the polymer (e.g. foracrylonitrile butadiene styrene (ABS), at 230° C.) and then setting thebuild platform to the glass transition temperature (e.g. for ABS 110°C.). At this low temperature extrusion, the material used for printingshould be able to can flow but still set up in a relatively short timeframe. The composition in accordance with certain exemplary embodimentscan set up in less than 2 minutes.

Current bioprinter inks that utilize cold extrusion have limitedstrength and adhesion to previous layers without cellular culture. Cellsare commonly suspended within the matrix and eventually grow and replacethe matrix. As noted, however, compositions used in accordance withexemplary embodiments does not require the use of cells, but has theability to include them due to the low melt flow temperatures.

The composition, although referenced as a bio-ink in relation to certainexemplary embodiments, need not be liquid in every case and may also besemi-solid or solid.

In some embodiments, the present invention provides compositions ofspecific materials of construction that promote cell proliferation inthe absence of any trophic agent using a base polymer poly(glycerol-sebacate) (PGS) as a bulk vehicle that may be co-modified withOGS, which can aid as a plasticizer, dispersing agent, wetting agent,processing aid or resin stabilizer.

In some embodiments of composition 12, various glycerol-sebacic acidesters and collagen and its derivatives as formulation modifiers areutilized. An advantage of this composition is uniformity of polymercomposition, extrusion at physiological temperatures, and instantaneoussolidification resulting in a fully bioresorbable polymer tissuescaffold that is comprised of biocompatible materials. It is a furtheradvantage of this composition that it is comprised of naturalmetabolites to accomplish formulating chemistry requirements.

The present invention may include a composition of matter 12 used as 3-Dscaffold building material, a.k.a. “bio-ink,” providing bioresorbableproperties with or without specific biological cells or trophiccomponents thereof, capable of being spatially deposited through a 3-Dprint nozzle 16 at physiological temperatures. The composition 12 may beformulated to go through a cold extrusion, via a syringe and plungersystem, material reservoir and pneumatic system or other extrusionthrough an orifice without the addition of heat. In one embodiment, thepresent invention provides composition 12 comprising water, apolypeptide, and a condensation polymer of a polyol and a diacid. Inanother embodiment, the composition 12 further comprises condensationoligomer of a polyol and a diacid. In another embodiment, a composition12 of the invention further comprises a bioactive material. In anotherembodiment of this invention condensation polymer and/or oligomer of thecomposition 12 can be functionalized to provide cross-linkable chemistryin the post print structure (i.e. the structure formed by printing andbefore further processing, also referred to as the “A stage” structure).Such functional groups may be induced to cause cross-linking, forexample by heating or photocuring the A stage structure, to provide forpost print stability (resulting in what may be referred to as a “Bstage” structure).

As discussed previously, in some embodiments composition 12 compriseswater; polypeptide, and an ester compound that is the condensationreaction product of a polyol and a diacid, preferably of glycerol andsebacic acid. In some embodiments, the polymeric glycerol-sebacic acidester and/or oligomeric glycerol-sebacic acid ester may befunctionalized. In some embodiments the polypeptide is collagen orgelatin. The relative amounts of the components of composition 12 may beadjusted to fine tune the physical properties of composition 12 and/orthe 3-D solid 10 built from composition 12.

In one embodiment, a composition in accordance with an exemplaryembodiment comprises

about 30% to about 85% by weight water;

about 10% to about 60% by weight glycerol-sebacic acid ester compound;and

about 0.1% to about 30% by weight polypeptide.

In another embodiment, the composition comprises

about 40% to about 85% by weight water;

about 10% to about 50% by weight glycerol-sebacic acid ester compound;and

about 5% to about 30% by weight polypeptide.

It will be appreciated that the specific amounts and ranges of amountsmay depend somewhat on the application for which the composition isemployed, with lesser amounts of polypeptide presently preferred forfiber applications.

In some embodiments the water component is present in a range of about35% to about 85% by weight. In some embodiments the water component ispresent in a range of from about 40% to about 80% by weight; such asabout 50% to about 75% by weight. In some embodiments the composition 12comprises about 40%, about 45%, about 50%, about 55%, about 60%, about65%, about 70%, about 75%, about 80%, or about 85% by weight water orany number, range or sub-range between any of the foregoing.

In some embodiments the glycerol-sebacic acid ester compound is presentas from about 10% to about 60% by weight, such as about 10%, about 15%,about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about50%, about 55%, or about 60% by weight, or any number, range orsub-range between any of the foregoing. The glycerol-sebacic acid estercompound may be present as PGS, OGS, or as a combination of the two.

In some embodiments the polypeptide component is present in a range ofabout 0.1% to about 30% by weight. In some embodiments, composition 12comprises about 0.5%, about 1%, about 5%, about 10%, about 15%, about20%, about 25%, or about 30%, by weight polypeptide as well as anynumber, range or sub-range between any of the foregoing.

In some embodiments, composition 12 comprises total solids in a range ofabout 15% to about 65% by weight.

In accordance with the present invention, poly-, polymer, and polymericrefer to intermediate weight polymers formed by esterification of apolyol and a diacid, oligo-, oligomer, and oligomeric refer to lowmolecular weight polymers formed by esterification of a polyol and adiacid, and thermoset or cured polymer refers to a high molecular weightpolymer formed by esterification of a polyol and a diacid and furthercrosslinked by heating, photocuring, microwave curing, infrared curingand the like. Condensation oligomers and polymers of a polyol and adiacid may also be characterized by the acid number (a measure of thenumber of carboxylic acid groups) and hydroxyl number (a measure of thenumber of hydroxyl groups). Acid number refers to mass of potassiumhydroxide (KOH) in milligrams that is required to neutralize one gram ofcondensation polymer or oligomer. Hydroxyl number refers to number ofmilligrams of potassium hydroxide required to neutralize the acetic acidtaken up on acetylation of one gram of condensation polymer or oligomer.Condensation oligomers useful in the invention typically have an acidnumber between about 50 mg/g and about 100 mg/g, about 55 mg/g and about85 mg/g, about 55 mg/g and about 75 mg/g, or about 55 mg/g and about 65mg/g; condensation polymers useful in the invention typically have anacid number between about 5 mg/g and about 55 mg/g, about 15 mg/g andabout 50 mg/g, about 20 mg/g and about 50 mg/g, or about 35 mg/g andabout 50 mg/g.

In some embodiments the polyol component may be glycol, glycerol,erythritol, threitol, arabitol, xylitol, mannitol, sorbitol, maltitol,or combinations thereof. In some embodiments the diacid may be sebacicacid, malonic acid, succinic acid, glutaric acid (5 carbons), adipicacid (6 carbons) pimelic acid (7 carbons), suberic acid (8 carbons), andazelaic acid (9 carbons). Exemplary long chain diacids include diacidshaving more than 10, more than 15, more than 20, and more than 25 carbonatoms. Non-aliphatic diacids may be used. For example, versions of theabove diacids having one or more double bonds may be employed to produceglycerol-diacid co-polymers. Amines and aromatic groups may also beincorporated into the carbon chain. Exemplary aromatic diacids includeterephthalic acid and carboxyphenoxypropane. The diacids may alsoinclude substituents as well. Reactive groups like amine and hydroxylmay increase the number of sites available for cross-linking Amino acidsand other biomolecules may modify the biological properties of thepolymer. Aromatic groups, aliphatic groups, and halogen atoms may modifythe inter-chain interactions within the polymer. Any condensationpolymer formed from of any of the above listed or other polyols and anyof the above listed or other diacids may be included in compositions ofthe invention.

Examples of poly(polyol sebacate)s for use in the present inventioninclude, but are not limited to, one or more of the following:poly(glycol-sebacate), poly(glycerol-sebacate),poly(erythritol-sebacate), poly(threitol-sebacate),poly(arabitol-sebacate), poly(xylitol-sebacate),poly(mannitol-sebacate), poly(sorbitol-sebacate),poly(maltitol-sebacate), and combinations thereof. In certainembodiments the poly(polyol-sebacate) is poly(glycerol-sebacate).

Examples of oligo(polyol-sebacate)s for use in the present inventioninclude, but are not limited to, one or more of the following:oligo(glycol-sebacate), oligo(glycerol-sebacate),oligo(erythritol-sebacate), oligo(threitol-sebacate),oligo(arabitol-sebacate), oligo(xylitol-sebacate),oligo(mannitol-sebacate), oligo(sorbitol-sebacate),oligo(maltitol-sebacate), and combinations thereof. In certainembodiments the oligo(polyol-sebacate) is oligo(glycerol-sebacate).

While OGS and PGS are described throughout as exemplary condensationoligomers and polymers, it is contemplated that any condensationoligomer or polymer may be used or substituted depending upon the needsof the artisan.

With respect to the mole ratio of polyol monomer to diacid monomer in acondensation polymer used in the present invention, such a mole ratio istypically about 1:1, though other ratios are within the scope of theinvention. In some embodiments, the mole ratio of polyol monomer todiacid monomer can be about 1:0.8, about 1:1, about 1:1.2, about 1:1.5,about 1:2, about 1.3, about 1:4, or about 2:3.

In some embodiments, the compositions 12 of the present inventioninclude condensation polymer to condensation oligomer in a weight ratioof polymer:oligomer that is about 10:1, about 9.5:1, about 9:1, about8.5:1, about 8:1, about 7.5:1, about 7:1, about 6.5:1, about 6:1, about5.5:1, about 5:1, about 4.5:1, about 4:1, about 3.5:1, about 3:1, about2.5:1, about 2:1, about 1.5:1, about 1:1, about 1:1.5, about 1:2, about1:2.5, about 1:3, about 1:3.5, about 1:4, about 1:4.5, about 1:5, about1:5.5, about 1:6, about 1:6.5, about 1:7, about 1:7.5, about 1:8, about1:8.5, about 1:9, about 1:9.5, or about 1:10.

In some embodiments, the composition 12 may be manipulated based on theadvancement of glycerol sebacic acid esterification of OGS and/or PGS.For instance, depending on the degree of OGS polymerization, OGS may actas a surfactant, wetting agent, functionalized carrier, flow andleveling agent, diluent, plasticizer, resin stabilizer, or specializedprocessing aid. Additionally, depending on the degree of polymerizationof PGS the polymer may be prepared as a gel, elastomer, resinthermoplastic or resin thermoset. Advancement of esterification isdetermined by measurement of acid number and hydroxyl numbers calculatedby those skilled in the art.

In some embodiments, a combination of condensation polymer andcondensation oligomer is used in bio-ink compositions of the presentinvention. A condensation polymer typically has an acid number in arange of from about 35 mg/g to about 50 mg/g and in some embodiments isabout 30 mg/g, about 35 mg/g, about 40 mg/g, about 45 mg/g, about 50mg/g, or about 55 mg/g. A condensation oligomer typically has an acidnumber in a range of from about 55 mg/g to about 65 mg/g and in someembodiments is about 50 mg/g, about 55 mg/g, about 60 mg/g, about 65mg/g, or about 70 mg/g.

In some embodiments the condensation polymer and/or condensationoligomer are functionalized. Examples of functional groups include, butare not limited to, acrylate, peptides, cinnamate.

In some embodiments, the condensation polymers or oligomers, such asglycerol-sebacic acid ester resins, may have one or more functionalgroups that may be “tuned” for hydrophilicity and, in contrast,hydrophobicity. That is, the condensation polymer and/or oligomer may bechosen such that any functional groups modifying the structure impart adesired hydrophilicity or hydrophobicity to the polymerized resin.Therefore it is possible to create custom functionalized resins based onfree hydroxyl and free carboxyl chemistries. Such tuning provides formodification of the composition such that the polypeptide loading can bemaximized in the aqueous dispersion.

It is further an advantage of tunability to maximize high solids of afully bioresorbable polymeric structure through the balance ofhydrophilicity and hydrophobicity.

In one embodiment, the composition of the present invention includes oneor more polypeptides, such as collagen and gelatin. Gelatin orequivalent collagen can be derived from, for example, human, Piscean,jellyfish, porcine, equine or bovine. In a like manner, collagen can besubstituted with gelatin A or B. In some embodiments, collagen Type 1,Type 2, Type 3, Type 4, Type 5, Type 6, Type 7, Type 8, Type 9, Type 10,Type 11, Type 12, Type 13, Type 14, Type 15, Type 16, Type 17, Type 18,Type 19, Type 20, Type 21, Type 22, Type 23, Type 24, Type 25, Type 26,or Type 27 may be used. In some embodiments dispersing a polypeptide inwater before the addition of a poly(polyol sebacate) or oligo(polyolsebacate) may enhance the dispersion of the poly(polyol sebacate) oroligo(polyol sebacate) in solution. In some embodiments of composition12, inclusion of a polypeptide may cause the extruded composition to setmore rapidly than a composition that does not include a polypeptide.

A polypeptide may be included in compositions of the invention, thoughany biologically active tissue, and in particular connective tissue canbe used. In some embodiments a polyurethane can be included in the placeof a polypeptide.

In some embodiments compositions of the present invention furtherinclude a bioactive material. Such a bioactive material can be vitamin,such as vitamin E or C, for example, mineral, and/or include tocopherol,ascorbate, retinoic acid, or combinations thereof. Alternatively, thebioactive material can be cells, such as stem cells, progenitor cells,mesenchymal cells, trophic cells, somatic cells, or combinationsthereof. In other embodiments, the bioactive material may bebiologically active short peptide sequences, growth factors,proteoglycans, glycoproteins, glycosaminoglycans and polysaccharides,nutrients, cytokines, hormones, angiogenic factors, immunomodulatoryfactors, drugs, or combinations thereof.

In other embodiments, the bioactive material can be selected from growthor morphogenic factors, such as, for example, transforming growthfactor, insulin-like growth factor 1, platelet-derived growth factor,bone morphogenetic proteins (bmps); cytokines, such as, for example,interleukins, chemokines, macrophage chemoattractant factors,cytokine-induced neutrophil chemoattractants (gro-1), integral membraneproteins such as integrins and growth factor receptors; membraneassociated factors that promote growth and morphogenesis, such as, forexample, repulsive guidance molecules; cell attachment or adhesionproteins, such as, for example, fibronectin and chondronectin; hormones,such as, for example, growth hormone, insulin and thyroxine;pericellular matrix molecules, such as agrin, laminin, thrombospondin,tenascin, veriscan, perlecan, syndecan, small leucine-rich proteoglycansand fibromodulin; nutrients, such as, for example, glucose andglucosamine; nucleic acids, such as, for example, RNA and DNA;anti-neoplastic agents, such as, for example, methotrexate andaminopterin; anti-inflammatory agents, such as, for example, naproxensodium, salicylic acid, diclofenac and ibuprofen; enzymes, such as, forexample, phosphorylase, sulfatase and kinase; and metabolic inhibitors,such as, for example, RNAi, cycloheximide and steroids.

In some embodiments, compositions of the present invention include afatty acid. Such a fatty acid may be admixed with thepoly(polyol-sebacate), oligo(polyol-sebacate) and polypeptidecomponents, or in some embodiments the fatty acid is co-esterified withpolymeric polyol-sebacate or oligomeric polyol sebacate. Fatty acids foruse with the invention include, but are not limited to, eicosapentaenoicacid, docosahexaenoic acid, arachidic acid, gadoleic acid, arachidonicacid, butyric acid, caproic acid, caprylic acid, capric acid, lauricacid, myristic acid, palmitic acid, palmitoleic acid, stearic acid,oleic acid, vaccenic acid, linoleic acid, alpha-linolenic acid,gamma-linolenic acid, behenic acid, erucic acid, lignoceric acid,cerotic acid, myristoleic acid, sapienic acid, elaidic acid, vaccenicacid, linoeladic acid, and combinations thereof.

Compositions 12 of the invention may comprise various aqueousdispersible combinations of (glycerol-sebacate) esters combined withgelatin or collagen or other suitable extracellular matrix (ECM)connective tissue polypeptide, polysaccharide and combinations thereof,and optionally including other physiological acceptable biomaterials andsolvents at a low temperature (less than about 40° C.). Thesecompositions may be useful in building 3-D scaffolds for tissueengineered constructs by extrusion, printing, and molding.

Any suitable method of forming a condensation reaction product of apolyol and a diacid may be employed for producing the ester component ofthe composition 12. One particularly suitable method is the processdescribed in U.S. application Ser. No. 14/725,654 filed May 29, 2015which is hereby incorporated by reference in its entirety.

One method of producing the composition 12, in some embodiments,comprises adding a solid polypeptide flour (e.g., gelatin or collagen)to warm or room temperature water and dispersing under mixing to form apolypeptide hydrogel and adding solid or molten ester to the polypeptidehydrogel dispersion under mixing to form an ester/polypeptide hydrogel;mixing the ester/polypeptide hydrogel under low heat; and cooling theester/polypeptide composition. The polypeptide hydrogel formed may be aviscous fluid dispersion. In some embodiments the warm water may be at atemperature of about 25° C. to about 85° C., or about 25° C. to about75° C. or about 25° C. to about 65° C. or about 30° C. to about 55° C.or about 35° C. to about 50° C. or about 40° C. to about 45° C. In someembodiments the low heat may be a temperature of about 25° C. to about150° C., about 40° C. to about 135° C., about 55° C. to about 120° C.,or about 70° C. to about 115° C. In some embodiments the low heat may bea temperature of about 25° C. to about 40° C., about 40° C. to about 75°C., about 75° C. to about 100° C., about 100° C. to about 115° C., orabout 100° C. to about 125° C. The ester/polypeptide hydrogel is stable(i.e. does not separate) and in some embodiments liquefies at atemperature at or below about 40° C., or in a range of about 35° C. andabout 40° C., or in a range of about 38° C. to about 40° C. Upon coolingthe composition solidifies at room temperature into a composition havinga soft texture that is somewhat similar to that of cheesecake. Despitethe original aqueous temperature used to dissolve the polypeptide, thecomposition may be able to flow at temperatures about or below 40° C.

Typically, low shear mixing is used to combine the components incomposition 12. In some embodiments, shear blade, lightning mixer, orother high shear mixing can be used to disperse a higher ratio ofpolypeptide and ester (e.g. glycerol-sebacic acid ester) into the water.

The method and order of mixing is aids in obtaining or avoiding certainproperties which might be desirable depending upon application.Polypeptide hydration in water should be consistently carried out as theinitial step followed by adding the ester in molten form to the hydratedmixture to achieve proper distribution throughout the solution.

In another embodiment, a method of producing a composition comprisesmodifying polarity of low molecular weight glycerol-sebacic acid estervia degree of polymerization for aqueous dispersibility based onhydrophilicity and hydroxyl functionality. This approach may vary theorder of added components according to formulation needs. For example,in one embodiment a method of producing composition 12 may includeadding a solid polypeptide flour (gelatin or collagen) to warm water anddispersing under mixing power to form a polypeptide hydrogel; adding OGSto the polypeptide hydrogel and dispersing under mixing power to form anOGS/polypeptide hydrogel; and adding PGS to the OGS/polypeptide hydrogeldispersion under mixing power to form a PGS/OGS/polypeptide hydrogel;mixing the PGS/OGS/polypeptide hydrogel under low heat; and cooling thePGS/OGS/polypeptide composition.

In addition the polarity of PGS and OGS may be further modified byco-esterification with an omega-# fatty acid. This modification woulddecrease the polarity of the resulting composition by the addition ofnon-polar alkyl chains.

3-D structures of the invention upon extrudation and cooling, but beforeprocessing to induce cross-linking (i.e., A-stage) may have aconsistency similar to cheesecake that is rigid but spongy and generallycapable of returning to its original shape after slight deformation.Depending on the ratio of polypeptide/PGS in the composition used toform the 3-D structure, the viscosity and tackiness may vary, withviscosity decreasing and tackiness increasing with increased PGS. Thetackiness may increase as the ratio of OGS:gelatin or the ratio of PGS:gelatin or the ratio of (OGS and PGS):gelatin increases.

Composition 12 may be printed as an A-staged composition and furtheradvanced to B-stage, in which the cross-linking has been induced withinand between the deposited layers of composition 12. In one embodiment,an A-staged composition may be advanced to B-stage by condensationpolymerization. Composition 12 may be delivered in multiple layers inthe X-Y plane at some predetermined vertical angle in an ink jet likeprinting apparatus or similar device providing spatial deposition. Thedeposition in-plane is repeated such that the sequential compilation ofplanes builds a 3-D construct of interest. A method for forming athree-dimensional scaffold may include extruding a first two-dimensionallayer of a bio-ink composition onto a substrate, and building a secondtwo-dimensional layer of a bio-ink composition upon the firsttwo-dimensional layer in a third dimension, wherein the bio-inkcomposition comprises a polymeric glycerol-sebacate and a polypeptide.

One of the features of this invention is that the glycerol-sebacic acidesters/polypeptide composition provides a biopolymer scaffold materialcapable of being extruded through the print head at physiologicaltemperatures. This feature further allows the direct incorporation ofbioactive components including biologics, trophic agents and cells (i.e.stem, progenitor, mesychencimal, or somatic). The temperature at whichcompositions can be processed through a nozzle may provide anotheradvantage over lactide and glycolide compositions. The 3-D print headnozzle temperature of a typical lactide or glycolide as an extrudate is150° C. to 250° C. These temperatures may make it impossible toincorporate cells or biologics into lactide and glycolide compositionsfor simultaneous biopolymer/cell extrudates useful in 3-D scaffoldprinting. The poly(glycerol-sebacate)/polypeptide composition of thecomposition 12 may be extruded through a 3-D print head nozzle atphysiological temperatures (i.e. about 37° C. to about 40° C.). Ascompared to typical polymer extrusion temperatures (e.g. 150° C. to 250°C.), being able to extrude the composition 12 at physiologicaltemperatures allows inclusion of bioactive materials, which typicallybiodegrade at temperatures greater than about 40° C. to 50° C. Thebio-ink composition may be extruded at a temperature at or below about40° C., or in a range of about 35° C. and about 40° C., or in a range ofabout 38° C. to about 40° C.

A further benefit of the composition 12 is the rapid solidification ofthe bioelastomer biodegradable polymer extrudate allowing forthree-dimensional additive buildup of structures as well as rapidprocessing of molds.

Methods of forming a 3-D structure may further include a step of heatingthe three-dimensional scaffold. A heating step may induce cross-linkingbetween and among the layers of the bio-ink composition, causing the 3-Dscaffold to “set”. Typically, the three-dimensional structure is heatedto a temperature in the range of about 30° C. to about 120° C., such asabout 30° C., about 40° C., about 50° C., about 60° C., about 70° C.,about 80° C., about 90° C., about 100° C., about 110° C., about 120° C.or any temperature, range or sub-range between any of the foregoing.

Methods of forming a 3-D structure may further include a step ofphotocuring the bio-ink composition. In one embodiment, the A-stageglycerol-sebacic acid esters may be acrylated to create a free radicalphotocurable extrudate. In this composition, the scaffold may beengineered in 3-D and “photo-set” in the A-stage before processing inthe B-stage. This can enhance handling and storage

The A-stage composition may be epoxidized to create a cationicphotocureable extrudate. In this composition the scaffold can beengineered in 3-D and “photo-set” in the A-stage before processing inthe B-stage. This can enhance handling and storage.

The A-stage composition may include a free co-blended fatty acid likethe omega-# fatty acids wherein the 3-D structure can be air-cured forpost print setting.

In other embodiments such as molding and extrusion the entire structurecan be processed with IR curing, acetone wash, lyphophilization, etc.

Furthermore, any structures formed of the compositions described hereincan be air dried. They may then be rehydrated as desired and, ifuncured, may expand to greater than their original size, although cured,crosslinked structures return to their original shape and size.

As noted previously, in some embodiments, the PGS and OGS may be furtherco-esterified with omega-# fatty acid. Compositions containing theomega-series may be air cured at room temperature (e.g., about 25° C.),or slightly elevated temperature, for example, at about 30° C., about35° C., about 40° C., about 45° C., or about 50° C.

The OGS/PGS/polypeptide/water compositions may be formulated withbiocompatible organic and inorganic materials with high dipole momentsand microwave cured. In some such embodiments the PGS may serve as amaterial having a high dipole moment depending on the number of freehydroxyls and carbonyl groups present. In some embodiments a materialwith a high dipole moment is admixed as part of the bio-ink composition12. In other embodiments the PGS is functionalized with a moiety havinga high dipole moment. Typical high dipole moment materials includeglycerol, polyethylene glycol (PEG) and other materials having carbonylor hydroxy groups.

An unexpected desirable benefit of the aqueous phase is that the waterbehaves like a porogen when the 3-D structure is dehydrated from thefinished construct. This provides porosity to the 3-D construct uponaqueous evacuation of said 3-D composition. In one embodiment, water maybe removed from the 3-D structure through lyophilization, as seen inFIG. 2. Typically, the 3-D structure is lyophilized at a temperature ofabout 0° C. to about −160° C., about −20° C. to about −140° C., about−40° C. to about −120° C., or about −60° C. to about −100° C. In someembodiments the three-dimensional structure may be lyophilized at atemperature of about 0° C., about −10° C., about −20° C., about −30° C.,about −40° C., about −50° C., about −60° C., about −70° C., about −75°C., about −80° C. about −85° C., about −90° C., about −100° C., about−110° C., about −120° C., about −130° C., about −140° C., about −150°C., or about −160° C. Thus, traditional porogens, such as sodiumchloride, which can lead to pocking or scarring of the material and canleave salt in the structure, even after washing, are not required.

The average pore size of the 3-D object 10 may be in the range of about5 μm to about 80 μm, about 10 μm to about 70 μm, about 20 μm to about 60μm, or about 30 μm to about 50 μm. In some embodiments the average poresize after dehydration is less than about 30 μm, less than about 25 μm,or less than about 20 μm. In some embodiments the average pore size isabout the size of or smaller than white blood cells. In some embodimentsthe average pore size is small enough to prevent macrophages to enterthe pores.

Another unexpected benefit of this composition is the uniformity ofpolypeptide fibrous network that can support and reinforce said esterpolymer structure. For scaffold constructs, collagen may be an importantextracellular matrix (ECM) component. The composition 12 may provide fora uniform dispersion of collagen and like-polypeptide derivatives to beuniformly dispersed.

In some cases, compositions in accordance with exemplary embodiments maybe printed directly on a material to provide a PGS coating on thematerial. Such coatings may impart beneficial properties to thesubstrate material, such as increased tensile strength of a woventextile. In some embodiments, the substrate material to be coated is apolymer. Typical polymers may include polyether ether ketone (PEEK),polyglycolic acid (PGA), polylactic acid (PLA), poly lactic-co-glycolicacid (PLGA), polyethylene terephthalate (PET), polypropylene (PP), andcombinations thereof.

Returning to FIG. 1, the 3-D printer assembly 100 includes the printhead 14 having the nozzle 16 and the reservoir 18 for holdingcomposition 12 and is further shown as comprising a heating element 20.Printer assembly 100 may further comprise a scanner 28 for scanning anobject and/or a computer aided design program for designing the object.

The heating element 20 can be employed to precisely aid in therheological properties of the composition 12 for physiologicaltemperature control.

Referring to FIGS. 6A-6D, and FIGS. 7A-7C, the heating element 20 mayinclude a bore 40 for receiving the nozzle 16. The heating element 20may further include an opening 42, which may extend from the outersurface of the housing element and opens into bore 40. Opening 42 mayreceive a set screw (not shown) and is configured to allow the set screwto be in communication with the nozzle 16. Heating element 20 mayinclude a bore 44 for receiving a heater cartridge (not shown). Opening46 extends from the outer surface of the housing element to bore 44 forreceiving a set screw (not shown) and is configured to allow the setscrew to be in communication with heater cartridge. Heating element 20may include a bore 48 for receiving a thermocouple.

The heating element 20 may be matched to the composition 12 so that thecomposition is heated to the desired temperature. The heating element 20may also be configured to allow for higher percent solids to be extrudedby allowing slight material flow, between 25-40° C.

In some embodiments heating element 20 has different zones to allow forcooling one or more reservoirs while heating one or more otherreservoirs.

The build platform 24 may be temperature controlled. In one embodiment,the temperature may be maintained at less than about 25° C., as opposedto a hot build platform, which is normally set to the material's glasstransition temperature. The cooled platform 24 may allow elevated flowlevels and ensure rapid set up of extruded build material. In someembodiments, the platform 24 is cooled to a temperature range of about0° C. to about 25° C., about 0° C. to about 10° C., about 5° C. to about15° C., about 10° C. to about 20° C., or about 15° C. to about 25° C.The cooled platform 24 may mimic placing the substrate and/or 3-Dstructure in a refrigerator for a faster set time.

Referring to FIGS. 1, 4, 5, and 7, nozzle 16 may be designed to allow abio-ink composition 12 to be extruded through cold extrusion, via asyringe and plunger system, material reservoir and pneumatic system orother extrusion.

Referring to FIGS. 4A-4E, the nozzle 16 may include a tip 30. The nozzle16 may include a feed line 36 for a second material, such as a fibercore. Accordingly, the nozzle 16 may be configured to create asheath-core extrudate wherein the ink composition may surround the fibercore. Typical fibers include collagen, polyglycolic acid (PGA),polycaprolactone (PCL), polylactic acid (PLA) and any otherbioresorbable fiber configured to add additional structure and strengthto the final print. The fiber core may be at a temperature cooler thanthat of the polymeric bio-ink composition. Typically, the fiber core maybe cooled to a temperature of about 25° C., about 30° C., about 35° C.,or about 40° C., about 25° C. to about 40° C., about 25° C. to about 35°C., or about 25° C. to about 30° C. In some embodiments, the fiber coremay be cooled to a temperature of about 5° C., about 10° C., about 15°C., about 20° C., or about 25° C. less than composition 12. Referring toFIG. 4E, in some embodiments the nozzle may further comprise a reservoirconnector element 19, as well a pitch associated with the bore of thenozzle to that allow for the mixing of composition 12 as it is beingextruded.

Currently, standard art ignores the fill pattern of the threedimensional builds. Infill density can be manipulated in currentpractice to affect the weight and amount of material used, caring solelyfor the external architecture. Bioprinting has incorporated pores orother internal structures designed into the three dimensional print,essentially printing at a 100% infill. The proposed invention alsomanipulates the internal architecture of the build, providing layer bylayer pattern formulation. Incorporated with the fiber core, the buildwill exhibit different properties based on the pattern and layering ofthe sheath core.

Referring to FIGS. 5A and 5B, an embodiment of the nozzle may havemultiple feeds 36 to the tip 30 such that a single nozzle may mono-co-or multi-extrude compositions on demand; such feeds 36 may be positionedin different arrangements according to the needs of the artisan. FIGS.5A and 5B depict a nozzle having 3 feeds, which is typical, but a nozzlehaving any number of feeds, such as one, two, three, four, five or moreis included within the scope of the invention. Each feed may beconnected to a reservoir for holding the composition 12 or a componentto be added to the composition 12. One or more feeds may be temperaturecontrolled. For example, the composition 12 may be heated to a firsttemperature and a feed having a biologic may be maintained at a secondtemperature lower than the first temperature. Referring to FIG. 5C, anembodiment may have multiple reservoirs 18 that lead to a single feed36. The multiple reservoirs would then be mixed through the bore asdescribed previously with respect to FIG. 4E.

The present invention provides for the design of a nozzle configurationwherein the said nozzle has a self-contained heat sink to provide energysufficient to allow said compositions to flow through said nozzlewithout harming the living cells. Said nozzle can be designed into aprint head having multiple reservoirs 18, each containing a differentcomposition, raw material, agent of growth, cell culture,physiologically acceptable medium, various ECM proteins (e.g., collagen,fibronectin, laminin, elastin, and/or proteoglycans), basic nutrientssuch as sugars and amino acids, growth factors, antibiotics (to minimizecontamination).

The composition 12 may additionally comprise non-cellular materials thatprovide specific biomechanical properties that enable bioprinting, orgrowth promoter.

Referring to FIG. 1, the 3-D printer assembly may include one or morecomputers 26 having one or more processors and memory (e.g., one or morenonvolatile storage devices). In some embodiments, memory or computerreadable storage medium of memory stores programs, modules and datastructures, or a subset thereof for a processor to control and run thevarious systems and methods disclosed herein. In one embodiment, anon-transitory computer readable storage medium having stored thereoncomputer-executable instructions which, when executed by a processor,perform one or more of the methods disclosed herein.

The construct instructions are delivered via software commands.According to another aspect of this invention the solid filling of the3-D object is manipulated by customized software.

Referring to FIG. 1, the 3-D print assembly may further include ascanner 28. A scanner may be used to scan a patient's organ, bone, orother anatomy to provide on the size and dimension of the 3-D structure10 to be constructed using the 3-D print assembly. A typical scanner isa CT scanner, but any scanner capable of scanning the patient's organ,bone, or other anatomy in three dimensions may be used.

EXAMPLES

A composition, Formulation 1, was formed comprising 10% gelatin, 59%water and 31% PGS, all by weight. Mixing was completed with a magneticstir bar and a temperature controlled hot plate. A control, Control 1,was neat PGS (made at 120° C. for 48 hours under vacuum).

Using an oscillatory rheometer, a reverse temperature sweep is performedon both materials to monitor the changes in the storage modulus (G′) asa function of temperature. The test was conducted by holding thecomposition at 40° C. for 60 seconds and then lowering the temperatureto 25° C. and holding for 300 seconds. The results are shown graphicallyin FIG. 3.

In this example, a G′ of ˜10 Pa is indicative of a free flowing viscousliquid so at 40° C. both Formulation 1 and the control are behaving asliquids. The Formulation 1 composition experiences a drastic increase instorage modulus after the temperature decreases, while the control'sstorage modulus remains constant. The increase in G′ indicates theFormulation 1 composition is solidifying and becoming more solid-like inmorphology, resulting in a material suitable as a bio-ink.

In FIG. 3, the control continues to flow even after dropping thetemperature from 40 to 25° C. The composition of Formulation 1, on theother hand, flows at 40° C. and then levels off completely after 100seconds at 25° C. The inconsistencies seen at 40° C. for both thecontrol and the Formulation 1, as well as the fluctuations of thecontrol held at 25° C. are indicative of the parallel plate torquedetection at or below the detection limit due to the low viscosity ofthe materials.

Since the control keeps this inconsistency throughout the test, it canbe assumed the 15° C. temperature decrease has no effect on the flow ofthe control. A gel modifier, like gelatin or collagen, such as is usedin Formulation 1, provides an ability to extrude the PGS containingcomposition and quickens the solidification process.

A composition of Formulation 2 was made of 13% gelatin, 74% water and13% PGS by weight. A composition of Formulation 3 was made of 11.5%gelatin, 65.5% water and 23% PGS by weight. Formulation 4 was made of10% gelatin, 59% water and 31% PGS by weight and Formulation 5 was 9.5%gelatin, 53% water and 37.5% PGS by weight. Each of the compositions inthese four examples were formulated with PGS made at 130° C. for 25hours under vacuum.

FIG. 12 shows several embodiments tested using similar methodology asdescribed in FIG. 3, except that the materials were held at 40° C. for240 seconds and 25° C. for 420 seconds. All four samples show the samecharacteristic solidification as described previously in FIG. 3 and thedegree of solidification is tunable based on the level of PGS present inthe formulation. The storage modulus increases (higher degree ofsolidification) with decreasing PGS concentration. Formulation 5 shows apreferred embodiment having rapid set time from a liquid phase to asolid phase, whereas Formulations 1-4 did not demonstrate a liquidphase.

In another example, a formulation was created to demonstrate theflexibility to be formed through an extrusion process. A 0.6% collagen,49.4% water and 50% PGS w/w formulation was extruded through a 480 μmorifice into an acetone processing bath. The striations of a purecollagen fiber (FIG. 9A) are similar to the striations seen in theexample (FIG. 9B) indicating the microstructure is similar. This processis capable of creating fibers with measurable tenacity.

In another example, the benefit of using low molecular weight ester as amodifier was demonstrated in the creation of a porous construct. Aformulation of 9.5% gelatin, 53% water, 18.75% low MW glycerol-sebacicacid ester (OGS), and 18.75% high MW glycerol-sebacic acid ester (PGS)was dehydrated in a processing bath and then thermally crosslinked for72 hours at 30° C. under vacuum. FIG. 10 is a cross sectional SEM imageof the resulting product and reveals a highly porous network with poresless than 50 μm. Without being bound by a specific theory, it isbelieved that the OGS acts as a porogen due to the increased solubilityof the material in acetone.

Compositions of Formulation 2 were formed by varying the order ofmixing. In one example, shear mixing of water, gelatin and molten PGSwas performed. In another example, shear mixing of gelatin added towarmed water was performed and then shear mixing of the water/gelatinmixture and molten PGS was performed. The compositions were then moldedand dehydrated and the results are shown in FIGS. 11A and 11B,respectively. FIG. 11A contains clear pockets of PGS that are not evenlydistributed, while FIG. 11B is evenly distributed with uniform porosity.

Another embodiment of the current invention is the ability to loadtherapeutic additives to the polymer/polypeptide composition. Acomposition similar to Formulation 4 was made consisting of 9.5%gelatin, 53% water, 28% PGS and 9.5% hydroxyapatite. Elemental mappingshowed the homogenous distribution of hydroxyapatite throughout theentire polymer/polypeptide composition.

It will be appreciated by those skilled in the art that changes could bemade to the exemplary embodiments shown and described above withoutdeparting from the broad inventive concepts thereof. It is understood,therefore, that this invention is not limited to the exemplaryembodiments shown and described, but it is intended to covermodifications within the spirit and scope of the present invention asdefined by the claims. For example, specific features of the exemplaryembodiments may or may not be part of the claimed invention and variousfeatures of the disclosed embodiments may be combined. Unlessspecifically set forth herein, the terms “a”, “an” and “the” are notlimited to one element but instead should be read as meaning “at leastone”.

It is to be understood that at least some of the figures anddescriptions of the invention have been simplified to focus on elementsthat are relevant for a clear understanding of the invention, whileeliminating, for purposes of clarity, other elements that those ofordinary skill in the art will appreciate may also comprise a portion ofthe invention. However, because such elements are well known in the art,and because they do not necessarily facilitate a better understanding ofthe invention, a description of such elements is not provided herein.

Further, to the extent that the methods of the present invention do notrely on the particular order of steps set forth herein, the particularorder of the steps should not be construed as limitation on the claims.Any claims directed to the methods of the present invention should notbe limited to the performance of their steps in the order written, andone skilled in the art can readily appreciate that the steps may bevaried and still remain within the spirit and scope of the presentinvention.

What is claimed is:
 1. A composition comprising: water; an ester of apolyol and a diacid; and a polypeptide.
 2. The composition according toclaim 1, wherein the ester comprises a glyercol-sebacic acid estercompound.
 3. The composition according to claim 2, wherein theglyercol-sebacic acid ester compound is a polymeric compound having amolecular weight greater than 10,000.
 4. The composition according toclaim 3, further comprising a glyercol-sebacic acid ester compoundhaving a molecular weight less than 10,000.
 5. The composition accordingto claim 2, wherein the composition comprises: about 30 to about 85% byweight water; about 10% to about 60% of the glyercol-sebacic acid estercompound; and about 0.1% to about 30% polypeptide.
 6. The compositionaccording to claim 2, wherein the composition comprises: about 40 toabout 85% by weight water; about 10% to about 50% of theglyercol-sebacic acid ester compound; and about 5% to about 30%polypeptide.
 7. The composition according to claim 1, wherein thepolypeptide is dispersed uniformly throughout the composition.
 8. Thecomposition according to claim 1, wherein the ester is functionalized.9. The composition according to claim 1, further comprising a bioactivematerial.
 10. The composition of claim 9, wherein the bioactive materialis selected from the group consisting of vitamins, minerals, tocopherol,ascorbate, retinoic acid, and combinations thereof.
 11. The compositionof claim 9, wherein the bioactive material comprises cells.
 12. Thecomposition of claim 9, wherein the bioactive material is selected fromthe group consisting of peptide sequences, growth factors,proteoglycans, glycoproteins, glycosaminoglycans, polysaccharides,nutrients, cytokines, hormones, angiogenic factors, immunomodulatoryfactors, drugs, and combinations thereof.
 13. The composition accordingto claim 1, further comprising a fatty acid.
 14. The compositionaccording to claim 13, wherein the fatty acid is co-esterified with thepolyol-diacid ester.
 15. The composition according to claim 1, whereinthe polypeptide is collagen
 16. The composition according to claim 1,wherein the polypeptide is gelatin.
 17. The composition according toclaim 1, wherein the composition consists of 8-12% by weight gelatin,35-40% by weight glyercol-sebacic acid ester compound, and the balancewater.
 18. The composition according to claim 1, wherein the diacid issebacic acid.
 19. A method for forming a composition comprising: mixinga solid polypeptide in water to form a polypeptide hydrogel; and addinga glyercol-sebacic acid ester compound to the polypeptide hydrogel. 20.The method of claim 19, wherein the step of adding the glyercol-sebacicacid ester compound is carried out while the glyercol-sebacic acid estercompound is in molten form.
 21. The method of claim 19, wherein theglyercol-sebacic acid ester compound is a polymeric compound having amolecular weight in excess of 10,000.
 22. The method of claim 20,further comprising mixing a glyercol-sebacic acid ester compound havinga molecular weight less than 10,000 into the polypeptide hydrogel.
 23. Amethod for printing a three-dimensional article comprising: extruding afirst two-dimensional layer of a composition according to claim 1 onto asubstrate; and building a second two-dimensional layer of thecomposition according to claim 1 upon the first two-dimensional layer ina third dimension.
 24. The method according to claim 23 furthercomprising curing the composition after the steps of extruding andbuilding.
 25. The method of claim 24, wherein the step of curingcomprises photocuring, microwave curing, infrared curing andcombinations thereof.
 26. A method for forming an article comprising:extruding a fiber of a composition according to claim
 1. 27. The methodaccording to claim 26, wherein the step of extruding comprisesco-extruding the composition of claim 1 with a second compositioncomprising a polymeric material.
 28. The method of claim 26, wherein thepolypeptide in the composition is collagen.
 29. A print head for use inadditive manufacturing comprising a nozzle and a plurality ofreservoirs, each reservoir containing a biocompatible material, thenozzle comprising a tip in fluid communication with each of thereservoirs via a feed line, the nozzle comprising a tip in fluidcommunication with each of the reservoirs via a feed line, the nozzlehaving a pitched bore to accomplish mixing of the biocompatiblematerials during extrusion.